Name: quantitative analysis of climbing defects in a drosophila model of neurodegenerative disorders
Uploaded: 2015-06-13T15:00:00
Description: Watch this Scientific Journal Video about Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders at JoVE.com

This work is supported by a Canadian Health Research Institute (CIHR) Team Grant to Dr. Bolduc (co-PI) and Dr. Guy Rouleau (PI). We would like to thank Dr. Oksana Suchowersky, Dr. Kathryn Todd, the members of the University of Alberta fly club and Dr. Clayton Dickson for help in development of the method and statistical analysis.

Research on Drosophila melanogaster was in compliance with the University of Alberta&#8217;s research guidelines.
1. Fly Collection
<ol>
	<li>Collect 20 flies using CO2 (g) anesthetization and place in a 25 mm x 95 mm collection vial containing food.</li>
	<li>Store vials containing flies horizontally to avoid trapping flies in any liquids that may accumulate in the bottom the vial.</li>
	<li>Incubate flies for at least 21 hr&#160;at 22 &#176;C at 45% humidity in an incub...

<ol>
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Drosophila has already proven to be an excellent model in Parkinson&#8217;s disease14 and other neurodegenerative conditions1,2. In addition to the genetic tools available in Drosophila, its genome is highly conserved for genes involved in neurological disorders19. The advent of genome wide genetic screening methods (including whole exome sequencing) is likely to continue to provide a larger list of candidate genes associated with human movement disorders. The developmen...

Devastating neurodegenerative disorders such as Parkinson&#8217;s disease, amyotrophic lateral sclerosis, and hereditary spastic paraplegia are increasingly recognized. Unfortunately, most of these neurodegenerative disorders are still without treatments. The widespread clinical use of genome-wide, unbiased genetic tests such as whole exome sequencing has led to an increasing number of genes being implicated in human locomotive disorders. Despite this progress, the pathological progression from early to late stages, remains elusive in these disorders. Drosophila provides one with the genetic tools for studying gene requirement in a controlled spatial and temporal manner. In addition, Drosophila has proven useful in screening drugs for neurological conditions such as Parkinson&#8217;s1, Alzheimer&#8217;s2, intellectual disability3,4 and epilepsy5,6 among others. Our aim was to develop a cost effective and reliable assay that would allow high throughput analysis that would still be sensitive enough to detect small changes in motor performance.
There are several assays used to quantify the effects of genetic mutation and/or environmental condition on Drosophila climbing behavior. Most of the assays capitalize on the natural tendency of flies to climb, known as negative geotaxis, or the climbing assay. Benzer7 suggested in 1967 that the counter-current apparatus used for the study of phototaxis could also be used to study gravitaxis. Since then, Ganetsky8 and many others9-12 have built on the initial assay. The principle is to place a known number of flies in a vial and tap the vial strongly against a hard surface, causing the flies to fall to the bottom of the vial. As it is an innate behavior, the flies will attempt to climb to the top of the vial, opposed to gravity. This assay is quantitative and measures how many flies have climbed past a marker on the vial during an allotted time period. Measurement of speed instead of total number of flies climbing has become a reliable parameter and shown defects in cases where the number of flies criteria was not significant13.
The climbing assay has proven useful in the study of many neurodegenerative disorders including Parkinson&#8217;s disease14. However, we noted that locomotive defects may not be detectable at time where neurodegeneration is already seen in pathological studies14. Thus, use of the traditional assay may limit the ability to study the early stages of disease pathogenesis. The appearance of locomotive defects during later stages of pathology may reflect a disease whose progression is too advanced for complete rescue.
This raises a potential issue with the sensitivity of the traditional climbing assay. The potential inability of the traditional climbing assay to detect mild locomotive defects can be attributed to the height to which the flies are required to climb. The traditional assay15,16 measures the number of flies to successfully climb over a height of 2 to 5 cm in 10 to 20 sec.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Drosophila stocks</td>
			<td></td>
			<td></td>
			<td>The stocks are selected depending on the experiments. The temperature and humidity in the room and in the incubator must be controled and consistent to avoid flies being too staticky or too wet.</td>
		</tr>
		<tr>
			<td>Video camera</td>
			<td></td>
			<td></td>
			<td>Any digital camcorder will do. Make sure they can focus on close object.</td>
		</tr>
		<tr>
			<td>Graduated cylinder</td>
			<td>Kimble</td>
			<td>20028W</td>
			<td>Different models of graduated cylinder may have different diameter. It is therefore imporant to measure the height.</td>
		</tr>
		<tr>
			<td>computer</td>
			<td></td>
			<td></td>
			<td>Any model will do. We used the computer to monitor the climbing of the flies and record the number of flies at each time point</td>
		</tr>
	</tbody>
</table>

quantitative analysis of climbing defects in a drosophila model of neurodegenerative disorders

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and thus current therapeutic treatment strategies are not curative. Through the use of whole exome sequencing, an increasing number of genes have been identified to play a role in human locomotion. Despite identifying these genes, it is not known how these genes are crucial to normal locomotive functioning. Therefore, a reliable assay, which utilizes model organisms to elucidate the role of these genes in order to identify novel targets of therapeutic interest, is needed more than ever. We have designed a sensitized version of the negative geotaxis assay that allows for the detection of milder defects earlier and has the ability to evaluate these defects over time. The assay is performed in a glass graduated cylinder, which is sealed with a wax barrier film. By increasing the threshold distance to be climbed to 17.5 cm and increasing the experiment duration to 2 min&#160;we have observed a greater sensitivity in detecting mild mobility dysfunctions. The assay is cost effective and does not require extensive training to obtain highly reproducible results. This makes it an excellent technique for screening candidate drugs in Drosophila mutants with locomotion defects.

Climbing is a strong and reproducible behavior. Indeed, one day old wild-type flies reach the target distance climbing performance rapidly (25 - 30 sec). Mutant flies present a range of performance from mild (or delayed) to complete inability to climb to the target. We illustrate this here with two different mutant alleles. The first one is a severe allele of the gene spastin caused by a complete deletion of the spastin gene (spas5.75) 18. In this line (spas5.75 with TM6b) one day old fli...

Watch this Scientific Journal Video about Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders at JoVE.com

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

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